Throughout this application, various publications are referenced by Arabic number within brackets. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed therein.
The expression of both histocompatibility antigens and tumor associated antigens (TAAs) of tumor cells can be augmented by treatment with bioresponse modulators, such as interferon and tumor necrosis factor-.alpha., and phorbol ester tumor promoters, such as TPA [1,13,14,16,18,22,23,27,28]. Upregulation of additional cellular antigens can be induced to a similar extent in both normal and tumor cells by bioresponse modulators indicating that this effect is a general property of these compounds and not restricted to TAAs or cells of a specific histotype (for review see [1,23]). For example, various interferons have been shown to enhance the expression of histocompatibility antigens, cellular antigens and TAAs in breast carcinoma, central nervous system tumors, colon carcinoma and melanoma cells [21-23,27,28,31,38]. In addition, when administered to animals containing human tumor xenografts, recombinant human interferon augments the ability of excised tumors to bind monoclonal antibodies specific for TAAs [13,21,36,42]. The use of bioresponse modifiers for increasing the expression of TAAs by tumor cells may prove useful in reducing the antigenic heterogeneity in tumors in vivo and augmenting the ability of monoclonal antibodies to bind to tumors (for review see [1,23,25,26]).
TPA and recombinant human leukocyte (IFN-.alpha.), fibroblast (IFN-.beta.) and immune (IFN-.gamma.) interferons increase both the expression and shedding of the tumor associated antigen BCA 225 by T47D cells and MCF-7 human breast carcinoma cells [36]. These compounds also increase the expression of the TAA carcinoembryonic antigen (CEA) and HLA Class II-DR antigen in both T47D and MCF-7 cells [20,36]. The mechanism by which TPA induces its diversity of effects in target cells is believed to be mediated initially by its binding to the Ca.sup.2+ -activated and phospholipid-dependent enzyme PKC which is the high affinity receptor for TPA (for review see [10, 43,44]). As a consequence of activation of PKC many important biochemical processes are initiated in target cells, including both positive and negative feedback controls in signal transduction pathways (for review see [43,44]). Recent studies have implicated PKC activation in mediating both antiviral activity and specific gene regulatory changes induced by IFN-.alpha., IFN-.beta., and IFN-.gamma. [6,8,37,46,47,50] and for review see [9]. The purpose of the present study was to explore the possible relationship between PKC activation and antigen upregulation induced by phorbol esters and interferon. With this aim in mind applicants have determined the effect of the synthetic PKC-activator ADMB, the natural PKC activators TPA and MEZ and the combination of PKC-activators and the PKC-inhibitor H-7 on the antigenic phenotype of T47D cells. To determine if similar biochemical pathways are involved in the ability of IFN-.beta. and IFN-.gamma. to alter the antigenic phenotype of T47D cells, applicants have also evaluated the effect of H-7 on interferon upregulation of the same antigens in T47D cells.
The effect of a synthetic protein kinase C (PKC) activator 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB) and the natural PKC-activating tumor promoting agents 12-0-tetradecanoyl-phorbol-13-acetate (TPA) and mezerein (MEZ) on the antigenic phenotype of carcinoma cells was studied. All three agents increased the surface expression of the tumor associated antigen such as BCA 225 and also of various cellular antigens, including HLA Class II antigens, intercellular adhesion molecule-1 (ICAM-1) and c-cerbB-2. Expression of the same antigens was also upregulated to various extent in T47D cells by recombinant fibroblast (IFN-.beta.) and immune (IFN-.gamma.) interferon. Shedding of BCA 225 from T47D cells was induced by TPA, MEZ, IFN-.beta. and IFN-.gamma., whereas ADMB did not display this activity. The ability of ADMB, TPA and MEZ to modulate the antigenic phenotype of T47D cells appears to involve a PKC-mediated pathway, since the PKC inhibitor, H-7, eliminates antigenic modulation. In contrast, the ability of IFN-.beta. and IFN-.gamma. to enhance HLA Class II antigens, c-erbB-2 and ICAM-1 expression, was either unchanged or modestly reduced by simultaneous exposure to H-7. Analysis of steady-state mRNA levels for HLA Class I antigens, HLA Class II-DR.beta. antigen, ICAM-1, and c-erbB-2 indicated that the ability of H-7 to inhibit expression of these antigens in TPA-, MEZ-, and ADMB-treated cells was not a consequence of a reduction in the steady-state levels of mRNAs for these antigens. The results of the present investigation indicate that the biochemical pathways mediating enhanced antigenic expression in T47D cells induced by TPA, MEZ and the synthetic PKC activator ADMB are different than those induced by recombinant interferons. Furthermore, upregulation of antigenic expression in T47D cells can occur by both a PKC-dependent or a PKC-independent pathway.